A novel β-carotene-9,10′-oxygenase (ScBCO2) has been characterized from Saccharomyces cerevisiae ULI3 to convert β-carotene to β-apo-10′-carotenal, which is a precursor of the plant hormone strigolactone. The ScBCO2 enzyme was purified to homogeneity by ammonium sulfate precipitation, Q sepharose and Superdex-200 chromatography. The molecular mass of the enzyme was ~50 kDa by SDS-PAGE. The purified ScBCO2 enzyme displayed optimal activity at 45 °C and pH 8. Tween 20 (1%, w/v), Trition X-100 (1%, w/v), Mg2+ (5 mM), Zn2+ (5 mM), Cu2+ (5 mM), Ca2+ (5 mM) or DTT (5 mM) increased in the activity by 3, 7, 14, 17, 23, 26 and 27%, respectively. ScBCO2 only exhibited cleavage activity towards carotenoid substrates containing two β-ionone rings and its catalytic efficiency (kcat/Km) followed the order β-carotene > α-carotene > lutein. ScBCO2 could be used as a potential candidate for the enzymatic biotransformation of β-carotene to β-apo-10′-carotenal in biotechnological applications.